Summary: This project was designed to generate "veto" cells which eliminate cytolytic effector cells specific for MHC alloantigen. MHC class I Dd was fused to a potent signaling molecule, the zeta (z) chain of the TCR and expressed on lymphocytes in transgenic mice. Transgenic mice expressing high copy numbers of the Dd-zeta fusion molecule have markedly diminished numbers of CD4+ and CD8+ T cells and a reciprocally high percentage of CD3-CD5+ cells which are TCR-CD4-CD8- cells but which are of T cell lineage as they express Thy 1.1,CD44, and Ly6C. Interestingly they express supraphysiologic level of Dd. These cells appear similar to memory CD8+ CTL, not only by cell surface appearance, but also functionally, as they have abundant cytolytic activity which can be activated by 1) TCR hybridomas recognizing cognate peptide in the context of Dd 2) mAb to Dd, and 3)cellular recognition of Dd by spleen cells from FVB mice primed to Dd. These cells potently kill cells that bind to Dd such as EL-4 cells which bind Dd through cell surface Ly49A. The mechanism by which CTL activity is mediated is being investigated, but does not appear to depend on Fas ligand expression or TRAIL expression. Furthermore, these memory CTL like cells also potently block the devlopment of an anti-Dd CTL reponse by both naive and primed FVB lymphocytes. Thus, these CD3-CD5+ T lineage cells represent an ideal type of a "veto" cell in being able to diminish even potent memory Dd specific CTL responses, yet because they lack TCR, they also lack the ability to induce GVHD. Additional studies are underway in which primary CD8+ T cells will be transfected with Dd-z using a lentiviral vector. The veto activity of such cells will then be tested in an in vivo skin allograft rejection model. Mcfarland, H.I.,Hansal, SA, Morris, D, McVicar, D,Weissman, A. Love, P.,and AS Rosenberg. Signaling through MHC in Transgenic Mice Generates a Population of Memory Phenotype Cytotlytic Cells that Lack TCR. Submitted.